This invention relates to microfluidic devices, and more particularly, relates to an apparatus for and a method of coupling a microfluidic device to an electrospray or other interface of a mass spectrometer.
Glass microfluidic devices have shown their vast potential in the field of analytical chemistry in the last decade and enable a certain amount of separation and analysis to be carried out. However, to augment the analytical capabilities of microfluidic chip systems, it is necessary to couple these devices to other instruments, and in particular it is desirable to be able to couple them to mass spectrometers. Other uses for this type of connection include, but are not limited to, coupling of the microfluidic device to conventional Capillary Electrophoresis (CE) detectors, sample introduction to a device, automation of a device and interconnections between devices. In large part, these microfluidic devices find their greatest utility in high performance separations. Therefore, connections to the devices must have minimal dead volumes so that the efficiency of the system is not compromised.
The coupling of separation methods with mass spectrometry provides a powerful tool for rapid identification of target analytes present at picogram levels in biological matrices, and structural characterization of complex biomolecules ranging from small pharmaceuticals to complex antibodies. Furthermore, mass spectrometry using electrospray ionization (ESMS) has emerged as a sensitive technique in a number of applications including the sequencing of peptides comprising common or modified amino acids, and the analysis of short DNA oligomers. Further modification of ESMS has improved sensitivity substantially through the use of ionization techniques operating at sub-microliter flow rates, giving xcexcESMS. The flow rates used and potentials applied in xcexcESMS are compatible with CE, and this has led to development of CE-xcexcESMS instruments, capable of initial separations followed by mass spectral analysis. A drawback of this approach is the 15-40 min. seperation on times often required, which tends to underutilize the spectrometer.
Microchip technology has recently been applied to CE, generating an extremely powerful separation and sample pretreatment tool (chip-CE) with analysis time of a few seconds. Separations have been combined on-chip with sample dilution, derivatization, enzyme digestion, and a set of independent manifolds for separation have been integrated on to a single chip to give a form of multiplexed analysis. Thus, sample pretreatment can be automated within an integrated device, a feature which could offer significant advantages in sample preparation for mass spectrometry, particularly if the chip could be designed as an ion source within an ESMS system.
Mass spectrometry using electrospray ionization has emerged as a sensitive technique, providing peptide analysis in the low nanogram range for digested protein using sequence tags and data base searching (Mann, M., Wilm, M., Anal. Chem., 66, 4390-4399 (1994)). Sequence information can be obtained from tandem mass spectrometric analysis where a given multiply-charged precursor ion is selected by the first mass analyzer and the fragment ions resulting from collisional activation with a neutral target gas (e.g. Argon) are transmitted into the second mass analyzer. The product ion spectra are characterized by easily identifiable series of fragment ions, and can be interpreted in the absence of protein or DNA sequence. Even in situations where only partial sequence is obtained, the sequence tag plus the peptide molecular weight can be used to locate the peptide in a given protein or data base. This combined approach was recently presented for the characterization of proteins from silver-stained polyacrylamide gels (Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Anal. Chem., 68, 850-858 (1996)). Such advances have been facilitated by the introduction of micro-electrospray ionization operating in the low nL/min flow rate regime (Wilm. M. Mann, M., Int. J. Mass Spectrom. Ion Proc., 136, 167-180 (1994)). Although this mode of sample introduction does not require any prior analyte separation (e.g. Liquid chromatography or CE), the sensitivity of the micro-electrospray technique can be adversely affected by the presence of salts used in proteolytic digestion or by the simultaneous ionization of a large number of different peptides isolated from digestion or by the simultaneous ionization of a large number of different peptides isolated from these digests. In addition, the mass spectra of unseparated digests are further complicated by the appearance of multiply-protonated molecules (M+nH)n+ for each peptide, which significantly compromise interpretation if more than one peptide is initially present. The combination of a high resolution separation technique to micro-electrospray sources thus confers a unique advantage in situations where both sensitivity and selectivity are desired.
The production of stable ionization conditions from micro-electrospray sources requires critical adjustment of low liquid flow rate (10-300 nL/min), column diameter, and field strength at the micro-electrospray tip. Consequently, the coupling of separation techniques to micro-electrospray is best achieved using CE, which typically operates in a flow rate regime of less than 300 nL/min. Recent reports have demonstrated the applicability of the capillary electrophoresis-micro-electrospray mass spectrometry (CE-xcexcESMS) approach for peptides and protein digests (Wahl, J. H., Gale, D. C., Smith, R. D., J. Chromatogr, 659, 217-222 (1994); Kriger, M. S., Cook, K. D., Ramsey, R. S., Anal. Chem.; 67, 385-389 (1995); Kelly, J. F., Ramaley, L. R.,Thibault, P., Anal. Chem. 69, 51-60 (1997)). As a result of the high separation efficiencies obtainable with CE, analyses conducted using CE-xcexcESMS typically yield 20-100 femtomoles mass detection limits in full-mass scan acquisition mode and 100-200 femtomoles for tandem mass spectrometric analyses. This is a 10-fold enhancement of sensitivity compared to more conventional (i.e. non-micro) CE-ESMS interface, using a coaxial sheath design operating at flow rates of 2-10 xcexcL/min.
The limited sample volume used in CZE (2% of capillary volume), results in concentration detection limits of approximately 1 xcexcM, even at 20 femtomole mass detection limits. Improvement in sample loadings can be achieved using isotachophoretic preconcentration (Foret, F., Szoko, E., Karger, B. L., J. Chromatogr, 608, 3 (1992); Foret, F., Sustacek, V., Bocek, P., J. Microcol. Sep., 2, 127 (1990); Mazereeuw, M., Tjaden, U. R., Reinhoud, N.J., J. Chromatogr. Sc., 33, 686 (1995)). This approach was successfully applied to the analysis of paralytic shellfish poisoning toxins present at low nM concentration levels in contaminated shellfish tissues, and enabled the injection of up to 1 xcexcL on a single capillary arrangement (Locke, S. J., Thibault, P., Anal. Chem., 66 6436 (1994)). On-line trace enrichment can also be obtained by loading large volumes of sample using microcolumns containing adsorptive media, followed by elution or electromigration onto a CE column. A review of different chromatographic preconcentrators has been presented recently (Tomlinson, A. J., Guzman, N. A., Naylor, S., J. Cap. Elect., 6, 2247 (1995)). These methods provide satisfactory means to overcome many detection limit problems.
Capillary Electrophoresis is a well established method and provides a number of separation formats thus giving flexibility for the analysis of different biomolecules. It is well suited as a sample introduction device to a mass spectrometer (Banks, J. F., Recent Advances in Capillary Electrophoresis/Electrospray/Mass Spectrometry. Electrophoresis, 18, 1997; and Cai, J. and Henion, J. Capillary Electrophoresisxe2x80x94Mass Spectrometry.J. Of Chromatography A., 703, 1995) At the most recent High Performance Capillary Electrophoresis Conference in Orlando Fla., several research groups reported on their efforts to directly interface microfluidic devices to mass spectrometers (Ramsey, R. S. And Ramsey, J. M. New Developments in Microchip ESI Mass Spectrometry; Figeys, D. And Aebersold, R. Microfabricated Devices Coupled to an Ion Trap Mass Spectrometer for the Identification of Proteins; and Liu, H., Foret, F., Zhang, B., Felten, C., Jedrzejewski, P. And Karger, B. L. Development of Microfluidic Devices for High Throughput ESI/MS). These groups have demonstrated that it is possible to obtain an electrospray directly from the microfluidic device, but they did not demonstrate high efficiency separations. The inventors"" experience with electrospray directly from the edge of a device, as in these other proposals, has shown that the droplet formed on the face of the device is sufficiently large that high efficiency separations are not possible due to the large mixing volume.
The effect of dead volumes is to distort the peak shape and increase band broadening. The maximum separation efficiency that can be observed with a microfluidic CE system joined to a capillary is limited by four principal sources of band broadening, namely longitudinal diffusion and effects of both injection and detection volume as well as any additional dead volumes.
One proposal has demonstrated reasonable separations with a device that included a pneumatic nebulizer (Foret, F., Liu, H., Zhang, B. and Karger, B. L., Single and Multiple Channel Microdevices for Microanalysis by ESI/MS. HPCE, Orlando, Fla., Feb. 1-5, 1998).
This still relies on forming an electrospray plume from the edge of the device, but combines this with a pneumatic or gaseous flow to improve nebulization of the emerging droplet, thereby reducing the droplet size and assisting in volatilization. A built in sprayer on the end of the chip is apparently simple and advantageous. However, it is believed that this can never give the same performance as a tapered capillary tip. Such a capillary tip provides a smaller droplet size, thus less dead volume, and less band broadening. The length of the capillary can be changed to meet changing resolution needs since separation continues in the capillary. These combined devices would be able to exploit commercial micro electrospray interfaces, with independent control over the electrospray operating parameters.
The literature has reported several methods used to join capillaries to microfluidic devices but to date they have shortcomings. Figeys et al. have constructed a butt joint to the edge of the chip with the use of a piece of Teflon tubing glued to the edge of the device as a guide sleeve and mooring point for the capillary. The capillary was used as an electro-osmotic pump for the introduction of protein digests to a MS device (Figeys, D., Ning, Y. And Aebersold, R., Anal. Chem., 69, 1997, p. 3153-3160). This article gives information regarding the dead volume of the connection. It also acknowledged the presence of contamination that may have been due to the epoxy used to glue the capillary in position. This method also requires that the capillary and the channel be aligned to within a few microns and held in position by the glue, a difficult task at best. This type of connection has the additional shortcoming that it is not possible to directly examine the joint for the presence of debris, glue or dead volume. These problems render this joining technique impractical for most applications.
With silicon it is possible to form a connection with minimal dead volume. This was demonstrated by van der Moolen et al (van der Moolen, J. N., Poppe, H. And Smit, H. C., Anal Chem., 69, 1997, P. 4220-4225). The SEM images of the interface presented in their article showed a tight connection with no apparent dead volume. The device was intended for correlation CE and no investigations were made for presence of band broadening introduced from the joint. Furthermore, the silicon device was used as an injector and performed the separation on the capillary. Unfortunately it is not possible to chemically etch deep structures into glass while retaining flat surfaces suitable for joining to a capillary so that the silicon procedure used by Moolen is inappropriate for glass. The problem with silicon devices is their inability to sustain the high electric fields that glass devices exploit for rapid separations. Consequently, a method to make low dead volume connections to glass devices is still needed.
Instrumental modifications are required to improve the analytical performance of the CE-xcexcESMS interface in terms of ruggedness and speed of analysis. The present invention is based on the development of a compact and versatile, micromachined chip device to perform CE or other sample manipulation and then introduce the sample to a xcexcESMS system, giving a chip-CE-xcexcESMS hybrid system. The chips are thus an integral component of the electrospray ion source for the mass spectrometer, providing both sample treatment and ion source functions. The intent of the present invention is to develop a chip-ES interface which is easily manufactured, so that it can be made commercially at lower cost than current methods, and can increase utilization and sample throughput of rather powerful, but expensive instruments such as ESMS systems. This ES interface will be reusable, but readily replaced when required by the user.
Interfacing chips to xcexcESMS would greatly expand the potential of both CE and ESMS for biotechnological applications requiring faster analysis time, enhanced sensitivity and selectivity. On-chip separations will provide for sample clean-up and separation of components to prevent interference in the mass spectrum, with a substantial reduction in analysis time (less than 5 and typically under 2 minutes). Minute sample and reagent consumption with less solvent and salt introduction at the interface should also lead to increased performance and efficiency.
To address this need, the inventors have developed a method of connecting fused silica capillaries to microfluidic devices. Silica capillaries were chosen because electrophoretic separations begun on the device can continue on the capillary and silica is transparent over a wide wavelength range. The initial invention was to develop an interface to MS that exploits the common micro electrospray. The results of MS experiments are presented.
In accordance with the present invention, there is provided a method of joining a capillary tube to a microchip including at least one capillary channel that opens onto an edge surface of the microchip, the method comprising the steps of:
(1) drilling a flat-bottomed hole into the edge surface of the microchip, the hole being axially aligned with the channel; and
(2) inserting an end of a capillary tube into the hole, abutting the capillary tube against the flat bottom, and bonding the capillary tube to the microchip;
so as to minimize dead volume between said capillary tube and said capillary channel.
The method can include bonding the capillary tube to the microchip with an adhesive substance. Advantageously, the adhesive substance is applied from the exterior, by capillary action and is permitted only to enter to the end of the capillary tube, without flowing substantially into an area between the end of the capillary tube and the capillary channel in the microchip.
Preferably, the method includes filling at least the end of the channel adjacent the edge surface of the microchip with an adhesive substance, to prevent substantial penetration of glass chips into the capillary channel. Conveniently, the substance is capable of being removed either by heating or by dissolution with a solvent.
It is preferred to provide the capillary with a tapered capillary tip, for use as an electrospray source for a mass spectrometer.
The hole can be drilled to a depth in the range of 2 to 5 times the diameter of the hole.
Advantageously, the method includes mounting the microchip in a drill press, providing a drill bit in the drill press, lowering the drill bit until it is close to the edge surface of the microchip, and viewing the drill bit through a magnifying means to ascertain that the drill bit is aligned with the capillary channel.
More preferably,the microchip device is mounted in a bracket, and the bracket is mounted on a Z axis translation stage, for movement in a horizontal X-Y plane, and the Z axis translation stage is adjusted to bring the capillary channel into alignment with the drill bit, for example by viewing the relative location of the drill bit to the capillary channel through a jeweller""s loupe.
A combination device comprising:
(a) a microchip having at least one channel opening onto an edge surface thereon, and a hole extending from the edge surface into the microchip and axially aligned with the channel, the hole being flat-bottomed and having a larger cross-section than the cross-section of the capillary channel; and
(b) a capillary tube mounted in the hole, abutting the flat bottom, and bonded to the microchip.
The capillary is advantageously bonded in position by an adhesive substance, which does not extend to an area between the end surface of the capillary tube and the flat bottom of the hole.
Another aspect of the present invention provides a method of joining a capillary tube to microchip including at least one capillary channel that opens onto an edge surface of the microchip, the method comprising the steps of:
(a) filling the end of the channel adjacent the edge surface of the microchip with a substantially solid substance having a substantially low melting point, to prevent substantial penetration of glass chips into the capillary channel;
(b) drilling a hole into the edge surface of the microchip, the hole being aligned with the capillary channel;
(c) removing the substantially solid substance from the capillary channel; and
(d) inserting an end of a capillary tube into the hole and bonding the capillary tube to the microchip.
Another aspect of the present invention provides a method of joining a capillary tube to a microchip including at least one capillary channel that opens onto an edge surface of the microchip, the method comprising the steps of:
(a) providing a stream of fluid through the capillary channel such that a continuous flow of fluid out from the free end of the channel is produced;
(b) drilling a hole into the edge surface of the microchip, the hole being aligned with the capillary channel such that the flow of fluid flushes away drilling debris; and
(c) inserting an end of a capillary tube into the hole and bonding the capillary tube to the microchip.
In yet another aspect of the present invention, this device is provided in combination with a mass spectrometer.
It is to be appreciated that this invention is not limited to application with mass spectrometers. More generally, the method is applicable to any aspect of microfluidic technology in which it is desired to connect a capillary tube to a microchip containing capillary channels. Such a device can be used to carry out a wide variety of different analytical and other techniques. The capillary tube itself need not necessarily have a free end, but could conceivably be connected at both ends to microfluidic chips, so as to provide an inter-connection between them. The capillary tube enables a channel of any desired length to be provided, and can enable a variety of different processing to be carried out, e.g. by irradiation of the tube and/or detection of substances travelling through the tube.
A chip-ESMS interface also offers other advantages. Microfabrication allows for multiple sample treatment manifolds on a single chip, so that multiplexing of sample introduction into a single MS is feasible, thereby increasing throughput. Also, regular supply of mass calibration standards is possible via the chip, giving improved mass accuracy. Finally, a variety of more complex sample treatments, such as on-chip digestion of proteins or DNA can further automate sample preparation and introduction in the xcexcESMS.